Over 70 million people in the U.S. are estimated to suffer from alterations in sleep-wakefulness. Existing therapies are of limited utility in treating these disorders. The difficulties in devising more effective treatments stem, in large part, from the fact that there is no one sleep center in the brain. As a result, the biological function(s) of sleep have not been elucidated. The neuronal circuitry underlying sleep-wakefulness is beginning to be understood. The intracellular events associated with the activity of these neurons across sleep-wakefulness, however, are not well defined. Understanding cellular mechanisms that occur during sleep is key to the identification of biological function(s). Acetylcholine (ACh) is associated with sleep-wakefulness. Preliminary results examining the ACh synthesis enzyme, choline acetyltransferase (ChAT) are consistent with the hypothesis that sleep may play a role in maintaining neurotransmitter levels that are depleted during waking via changes in mRNA during sleep. The vesicular acetylcholine transporter (VAChT) performs the essential function of packaging nascent acetylcholine into vesicles. The gene encoding yacht lies within the gene encoding chat in all species examined, an extremely rare mammalian genomic organization. Preliminary studies suggest that chat and vacht mRNA levels are differentially expressed during sleep. In these studies, we focus first on delineating VAChT expression. In the first specific aim, we test the hypothesis that vacht mRNA and protein vary across individual sleep-wake bouts (i.e., waking, SWS and REM sleep) occurring over a 24h period. Preliminary studies of vacht mRNA suggest that it is higher during waking than during sleep. The second specific aim tests the hypothesis that mRNA and protein are associated with wakefulness. Initial results show vacht mRNA did not vary following six hours of sleep deprivation compared to controls. Specific aims 3 and 4 focus on comparisons of vacht and chat mRNAs, given the genomic organization of the genes and preliminary results that show that vacht mRNA contrasts with chat mRNA expression. Specific aim 3 tests the hypothesis that chat and vacht mRNAs are differentially expressed during sleep, while specific aim 4 tests the hypothesis that vacht mRNA subtypes are differentially expressed in response to sleep-wakefulness. Identification of these patterns of expression are key to understanding cellular mechanisms underlying sleep and wakefulness.